Directed evolution gets a significant speed boost

Synthetic biology attempts to generate biomolecules with new and/or improved activities for use as, say, drugs or fuel. One way of producing new biomolecules involves directed evolution, in which specific functions are selected for, but that has been slow and labor intensive. A faster method comes from Esvelt et al. at Harvard, who have devised a system for directed evolution that is rapid and continuous, cycling through twenty to thirty rounds of selection in a single day without human intervention.

The new system relies on phage, viruses that infect bacteria, and is thus called "phage-assisted continuous evolution"—yielding the apt acronym PACE. In this system, molecules from E. coli flow through an evocatively named "lagoon" filled with phage. Each phage contains a copy of the Gene Of Interest (GOI), the one that will undergo directed evolution.

Being viruses, the phage can’t replicate DNA on their own; they need to get into a bacterial cell to do that. In order to successfully infect a bacterium—to not only get into the E. coli cell, but to force it to make more phage and spew them out—the phage require a protein called pIII.

To force the GOI to evolve, the researchers took the gene for pIII out of the phage and put it into the bacteria, linking its expression to the activity of the GOI. Thus, phage containing more active versions of the GOI will generate more pIII, and will be more infectious, spreading more copies of that version of the GOI as they infect more hosts. Eventually, only the most successful mutant version(s) of the GOI will be left. To speed things up a bit, the bacteria are also given a "mutagenesis plasmid" that suppresses DNA proofreading, which increases the mutation rate.

The research team demonstrated PACE’s speed by evolving a number of variants of T7 RNA polymerase, a well studied enzyme responsible for transcribing DNA into RNA. T7 RNA polymerase binds to a very specific DNA sequence, the T7 promoter, and transcribes only those genes preceded by this sequence. Esvelt et al. rendered pIII production dependent on the protein binding to a different sequence.

After a week of PACE that encompassed 200 rounds of evolution, the team made T7 RNA polymerase that bound to the alternate promoter, sometimes with higher activity than it has on its native target.

Two different PACE experiments were carried out concurrently in separate lagoons, and although both eventually made the same mutant T7 RNA polymerase, they took different routes to get there. The scientists also made T7 RNA polymerase mutants that initiated transcription with ATP or CTP instead of the GTP it prefers; these took only 36 hours.

The authors note that “the PACE system can be assembled entirely from a modest collection of commercially available equipment and does not require the manufacture of any specialized components,” making it ideal for those DIY bio types hanging out in Brooklyn. In literally bringing directed evolution up to speed, PACE may also help answer questions about molecular evolution as it occurs in nature while generating new biomolecules with desirable activities and traits.